The transcription factor Nrf2 has emerged as a master regulator of a cellular protective mechanism by upregulating antioxidant response element (ARE)-bearing genes encoding antioxidant enzymes, detoxifying enzymes, xenobiotic transporters, and stress response proteins. Keap1, a substrate adaptor protein for a Cullin3 (Cul3)-based E3 ubiquitin ligase, tightly regulates the Nrf2-ARE signaling pathway. Under basal conditions, Nrf2 is constantly targeted for Keap1-mediated ubiquitination and subsequent proteasomal degradation to maintain a low constitutive level in al human organs. Upon activation of the pathway, the enzymatic activity of the Keap1-Cul3-Rbx1 E3 ligase is inhibited, resulting in stabilization of Nrf2 and activation of Nrf2 downstream genes. Since the discovery of the Nrf2-Keap1-ARE signaling pathway in 1999, Nrf2 has been viewed as a "good" transcription factor that protects us from oxidative stress-related diseases, including cancer. The chemopreventive property of Nrf2 has been well documented by the following two facts: (i) many of the well- studied chemopreventive compounds elicit their activities through activation of the Nrf2-ARE signaling pathway, and (ii) Nrf2-null mice are highly susceptible to chemical carcinogens and are no longer protected by chemopreventive compounds. Paradoxically, the "dark side" of Nrf2 has recently been revealed. For instance, somatic mutations that disrupt the Keap1-mediated negative regulation of Nrf2, resulting in a high constitutive level of Nrf2, have been identified in several types of tumors and cancer cel lines, especially non-small cell lung carcinoma (NSCLC). Furthermore, mounting evidence has emerged, indicating that Nrf2 contributes to chemoresistance, the major obstacle in cancer treatment. The discovery of the "dark side" of Nrf2 has clearly illustrated the urgent need to identify Nrf2 inhibitors and develop them into druggable compounds to enhance the efficacy of cancer treatments. We have screened a large number of natural products for their inhibition of ARE-luciferase activity using a stable cell line established in our lab, MDA-MB-231-ARE-Luc, containing an ARE (from GST-Ya)-dependent luciferase gene. Using this method, we have identified a plant extract that is able to inhibit ARE-luciferase activity. Furthermore, a pure compound, brusatol, has been isolated from the extract and has been found to inhibit the protein level of Nrf2 and exhibit potent anti-cancer activities. So far, we have obtained a substantial amount of preliminary data demonstrating that brusatol sensitizes several cancer cell lines to chemotherapeutic drugs in vitro and, more significantly, brusatol sensitizes lung cancer xenografts to cisplatin in vivo in an Nrf2-dependent manner. Based on the ability of brusatol to specifically inhibit Nrf2 and sensitize cultured cancer cells and xenografts to cisplatin treatments, we hypothesize that brusatol can enhance the efficacy of current cancer treatments by sensitizing cancer cells to chemotherapeutic drugs through inhibition of the Nrf2-dependant protective mechanism. The goal of the proposed research is to further characterize the anti-cancer properties of brusatol using a preclinical lung cancer model and delineate the molecular targets and mechanistic actions of brusatol. The proposed study will not only provide a framework for the development of this Nrf2 inhibitor into a therapeutic drug to combat chemoresistance, but also provide the first Nrf2 inhibitor for basic research in the field, both of which will have profound impacts on human health worldwide. Therefore, the following three aims will be pursued: Aim #1. Characterize the mechanistic actions of brusatol-mediated Nrf2 inhibition Nrf2 is primarily regulated by the Keap1-Cul3-Rbx1 E3 ligase at the protein level through ubiquitination and degradation. Therefore, we will investigate the effects of brusatol on the proteins that may enhance the activity of the E3 ligase, such as the protein subunits of the E3 ligase, as well as regulatory proteins that control the dynamic assembly/disassembly of the ligase complex. Aim #2. Determine the molecular targets of brusatol The target proteins of brusatol will be identified and verified. The biological functions of these proteins, in particular their crosstalk with the Nrf2 signaling pathway, wil be investigated. Most likely, these proteins will directly or indirectly regulate the Keap1-Cul3-Rbx1 E3 ligase. Aim #3. Evaluate the in vivo efficacy of brusatol using an LSL-KrasG12D/+ mouselung cancer model The feasibility of using brusatol as an adjuvant to enhance current cancer treatments and to combat both intrinsic and acquired resistance will be tested in this preclinical model that recapitulates the development and progression of human lung cancer. ! "! PUBLIC HEALTH RELEVANCE: Lung Cancer is the leading cause of cancer-related death worldwide. Little progress has been made in the treatment of lung cancer due to high resistance of lung cancer cells to chemotherapeutic treatments. Nrf2 is a protein that regulates one of the most important cellular defense mechanisms to cope with environmental toxins and chemotherapeutic drugs. Therefore, inhibition of Nrf2 represents a novel mechanism to sensitize cancer cells to chemotherapeutic drugs. This proposal focuses on the discovery and development of a phytochemical, brusatol, into a druggable compound that inhibits the Nrf2- mediated protective pathway, enhancing the effectiveness of a broad range of cancer treatments. In addition, mechanistic actions of brusatol will also be investigated.